- Email: [email protected]
A fluorescent stain for viable rosette-forming cells
Journal of Immunological Methods 5 (1974) 305-306. © North-Holland Publishing Company
A FLUORESCENT STAIN FOR VIABLE ROSETTEFORMING CELLS R. RAMASAMY Department of Pathology, Cambridge University, Tennis Court Road, Cambridge, U.K.
Received 4 March 1974
Accepted 25 April 1974
A rapid fluorescent staining technique for visualising viable rosette-forming cells is described.
1. INTRODUCTION The rosette test is widely used in immunology for determining surface immunoglobulin, Fc and C3 receptors, and in direct tests for antigen binding, on lymphoid cells. It is sometimes necessary during these tests to count only viable reacting cells since dead or dying cells give artefactual results. Conventional vital stains work by an exclusion principle i.e. living cells are left unstained. Celada and Rothman (1967) described a fluorescent stain for viable cells. I have shown here that the technique can be used to visualise viable cells within a rosette of erythrocytes.
2. MATERIALS AND METHODS A rosette test for detecting Fc receptors on murine lymphoid cells has been described (Ramasamy and Munro, 1974). Fc rosettes were formed on a non secreting variant plasmacytoma 289-16 maintained in tissue culture. (Ramasamy et al., 1974). A stock solution of diacetyl fluorescein (Koch Light Laboratories, Colnbrook, Bucks, England) at 5 mg/ml in acetone was diluted 1 ' 104 in phosphate-buffered saline immediately prior to use. One drop of staining solution was mixed with one drop of the rosetted suspension on a slide and viewed under incident U.V. lighting with a low transmitted light background, so that both fluorescent tumour cells and rosetting erythrocytes were simultaneously visible. Interference filters Balzer FITC 5 and G G 4 7 5 together with an OG 515 barrier filter were used. Photographs were taken with an Ilford EP FP 4 fdm. 305
306
R. RAMASAMY
Fig. 1. 289-16 Cells stained with diacetyl fluorescein in an Vc receptor test. 3. RESULTS AND DISCUSSION A photograph of a typical preparation is presented above (fig. 1). The technique described permits a rapid counting of rosette-forming preparations. There is a gradual increase in the background fluorescence of the medium with time. The preparations maintained at 4°C are countable up to 2 hr after staining. The size of the fluorescent cell can be seen through the rosette of erythrocytes and large cells e.g. macrophages and tumour cells can be distinguished from small lymphocytes in a mixture.
ACKNOWLEDGEMENTS This work was supported in part with a grant from the Cancer Research Campaign. I thank P. Curtis for the photography and the Wellcome Trust for a studentship.
REFERENCES Celada, G. and B. Rothman, 1967, Proc. Natl. Acad. Sci. U.S. 57,530. Ramasamy, R. and A.J. Munro, 1974, Immunology, 26,563. Ramasamy, R., A.J. Munro and C. Milstein, 1974, submitted to Nature.
A FLUORESCENT STAIN FOR VIABLE ROSETTEFORMING CELLS R. RAMASAMY Department of Pathology, Cambridge University, Tennis Court Road, Cambridge, U.K.
Received 4 March 1974
Accepted 25 April 1974
A rapid fluorescent staining technique for visualising viable rosette-forming cells is described.
1. INTRODUCTION The rosette test is widely used in immunology for determining surface immunoglobulin, Fc and C3 receptors, and in direct tests for antigen binding, on lymphoid cells. It is sometimes necessary during these tests to count only viable reacting cells since dead or dying cells give artefactual results. Conventional vital stains work by an exclusion principle i.e. living cells are left unstained. Celada and Rothman (1967) described a fluorescent stain for viable cells. I have shown here that the technique can be used to visualise viable cells within a rosette of erythrocytes.
2. MATERIALS AND METHODS A rosette test for detecting Fc receptors on murine lymphoid cells has been described (Ramasamy and Munro, 1974). Fc rosettes were formed on a non secreting variant plasmacytoma 289-16 maintained in tissue culture. (Ramasamy et al., 1974). A stock solution of diacetyl fluorescein (Koch Light Laboratories, Colnbrook, Bucks, England) at 5 mg/ml in acetone was diluted 1 ' 104 in phosphate-buffered saline immediately prior to use. One drop of staining solution was mixed with one drop of the rosetted suspension on a slide and viewed under incident U.V. lighting with a low transmitted light background, so that both fluorescent tumour cells and rosetting erythrocytes were simultaneously visible. Interference filters Balzer FITC 5 and G G 4 7 5 together with an OG 515 barrier filter were used. Photographs were taken with an Ilford EP FP 4 fdm. 305
306
R. RAMASAMY
Fig. 1. 289-16 Cells stained with diacetyl fluorescein in an Vc receptor test. 3. RESULTS AND DISCUSSION A photograph of a typical preparation is presented above (fig. 1). The technique described permits a rapid counting of rosette-forming preparations. There is a gradual increase in the background fluorescence of the medium with time. The preparations maintained at 4°C are countable up to 2 hr after staining. The size of the fluorescent cell can be seen through the rosette of erythrocytes and large cells e.g. macrophages and tumour cells can be distinguished from small lymphocytes in a mixture.
ACKNOWLEDGEMENTS This work was supported in part with a grant from the Cancer Research Campaign. I thank P. Curtis for the photography and the Wellcome Trust for a studentship.
REFERENCES Celada, G. and B. Rothman, 1967, Proc. Natl. Acad. Sci. U.S. 57,530. Ramasamy, R. and A.J. Munro, 1974, Immunology, 26,563. Ramasamy, R., A.J. Munro and C. Milstein, 1974, submitted to Nature.